Methylation is a common covalent modification of biological small molecules, nucleic acids, and proteins. Methylation is a prominent posttranslational modification in intracellular signaling pathways. In eukaryotes, genomic DNA and histones that comprise chromatin are subject to this modification. Methylation of cytosine bases in CpG dinucleotide repeats is enriched in transcriptionally repressed chromatin and mediates epigenetic silencing within these domains (Hermann et al., Cell Mol. Life Sci. 61 (2004) 2571-2587). Histones H3 and H4, which constitute part of the histone octamer in the nucleosome core particle, undergo methylation at distinct arginine and lysine residues (Trievel, Crit. Rev. Eukaryot. Gene Expr. 14 (2004) 147-170). In addition, linker histone H1b, which binds to the exterior of the nucleosome, is subject to methylation at Lys-26 (Kuzmichev et al., Mol. Cell 14 (2004) 183-193).
Efforts by numerous groups have identified many of the enzymes that methylate DNA and histones in chromatin. These enzymes all share a common mechanistic feature in that they utilize S-adenosylmethionine (AdoMet) as the methyl-donating cofactor. The enzymes that catalyze methylation of CpG repeats are generally known as DNA methyltransferases or DNMTS (Herman et al., supra), while the enzymes that methylate arginines and lysines in histones are collectively referred to as histone methyltransferases (HMTs). HMTs can be further classified based on their amino acid specificity: protein arginine methyltransferases (PRMTs) and histone lysine methyltransferases (HKMTs) (Trievel, 2004, supra). Methylation of specific arginines and lysines within core and linker histones by HMTs has been directly linked to transcriptional regulation. For example, methylation of Lys-4 in histone H3 is enriched in transcriptionally active loci, whereas H3 Lys-9 methylation is a hallmark of heterochromatin and silent euchromatin (Sims et al., Trends Genet. 19 (2003) 629-639). Moreover, several HKMTs have recently been implicated in DNA repair checkpoints in the cell cycle (Sanders et al., Cell 119 (2004) 603-614; Giannattasio et al., J. Biol. Chem. (2005)), suggesting that these enzymes may have broader roles in chromatin remodeling than previously believed.
Elucidating the determinants of the substrate specificity of HMTs is pivotal to understanding the biological functions of these enzymes. Unlike histone acetyltransferases (HATs), which are generally promiscuous with regard to their substrate specificity (Marmorstein, J. Mol. Biol. 311 (2001) 433-444), most HMTs are highly selective and site-specifically methylate discrete residues within histones. This selectivity is exemplified by HKMTs, many of which modify only individual lysyl residues within histones H1b, H3, or H4 (Trievel, 2004, supra, Kuzmichev et al., supra). To characterize the substrate specificities of these enzymes, radioactive methyltransferase assays, which measure the incorporation of tritiated methyl groups from radiolabeled AdoMet into protein or peptide substrates, have been used to determine sites of lysine methylation within histones and other nuclear proteins (Nishioka et al., Methods 31 (2003) 49-58). The steady state kinetic parameters of several HMTs have been quantitatively measured using this technique, including the human histone H3 Lys-4-specific methyltransferase SET7/9 (Trievel et al., Cell 111 (2002) 91-103) and the H3 Lys-9-specific enzymes mouse ESET (Wang et al., Mol. Cell 12 (2003) 475-487), Drosophila SU(VAR)3-9 (Eskeland et al., Biochemistry 43 (2004) 3740-3749), and mouse G9A (Patnaik et al., J Biol. Chem. (2004)). Although highly sensitive, the radiometric assay is laborious and not suited to high-throughput applications. Furthermore, the accumulation of AdoHcy during this assay can result in significant product inhibition of HMTs (Patnaik et al., supra, Kim et al., Cancer Res. 63 (2003) 7619-7623) and lead to errors in determining the steady state kinetic parameters of these enzymes.
What is needed are more efficient and accurate assays for HMT activity.